S. rimosus ATCC 10970 was obtained from China General Microbiological Culture Collection Center (CGMCC 4.1438). E. coli JM109 and BL21(DE3) were used for general cloning and protein expression, respectively. LB medium was used for E. coli cultivation. Kanamycin was used at a final concentration of 50 μg/mL. Restriction enzymes and Q5 DNA polymerase were purchased from New England Biolabs (United States). DNA manipulations, competent cell preparation, and transformation were performed as described previously (Sambrook and Russell, 2001 ). Hydrogen peroxide (30%) solution was purchased from Sigma-Aldrich. The 96-well UV-transparent microplates were purchased from Corning.
Hydrogen peroxide 30 solution
Manufactured by Merck Group
Sourced in United States
Hydrogen peroxide (30%) solution is a clear, colorless liquid chemical compound. It has the chemical formula H2O2 and is used as an oxidizing agent in various industrial and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Kanamycin, by New England Biolabs (1 mentions)
Q5 dna polymerase, by New England Biolabs (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Potassium thiocyanate, by Merck Group (1 mentions)
Potassium iodide, by Merck Group (1 mentions)
Sodium cyanide, by Merck Group (1 mentions)
Potassium bromide, by Merck Group (1 mentions)
Sodium ascorbate, by Merck Group (1 mentions)
Sodium hexachloroplatinate 4 hexahydrate, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Gold 3 chloride hydrate, by Merck Group (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride edc, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Hydroquinone, by Merck Group (1 mentions)
11 protocols using hydrogen peroxide 30 solution
1
Heterologous Expression of S. rimosus
2
Bovine Lactoperoxidase Activity Assay
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Bovine lactoperoxidase (L2005), sodium chloride, potassium thiocyanate, sodium cyanide, and hydrogen peroxide (30% solution) were purchased from Sigma. The concentration of hydrogen peroxide was determined at 240 nm using the molar extinction coefficient of 39.4 m −1 cm−1 (38 (link)). Potassium bromide and potassium iodide were obtained from Merck. All other chemicals, if not stated otherwise, were purchased from Sigma at the highest grade available. H2O2 solutions and potassium iodide were prepared fresh before use.
3
Immunochemical Labeling and Detection Protocol
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OA, soybean trypsin inhibitor (STI), bovine serum albumin (BSA), gold(III) chloride hydrate (HAuCl4·H2O), sodium hexachloroplatinate (IV) hexahydrate (Na2PtCl6·6H2O), sodium ascorbate, methanol, sucrose, dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS), Triton X-100, sodium azide, 3,3′-diaminobenzidine (DAB), hydrogen peroxide (30% solution), and N-(3-dimethyl aminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) were from Sigma-Aldrich (Saint Louis, MO, USA). A peroxidase substrate solution based on 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Immunotech (Moscow, Russia). Polyclonal goat anti-mouse immunoglobulins (GAMI) were from Arista Biologicals (Allentown, PA, USA), polyclonal donkey anti-goat immunoglobulins (DAGI), and GAMI labeled with horseradish peroxidase (GAMI–HRP) were from Jackson Immuno Research Labs (West Grove, PA, USA).
4
Electrochemical detection of Legionella pneumophila
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Different set of buffers were used for specific procedures in the experiments, and their composition is described in S1 (Supp. data). All buffers were prepared from chemicals of analytical grade purchased from Merck and Sigma and using milliQ water. The reagents used for the electrochemical measurement includes hydroquinone (Ref. H9003) and hydrogen peroxide 30% solution (Ref. 31642, Sigma-Aldrich).
The anti-L. pneumophila monoclonal antibody (G90A) from mouse (Catalogue no. MA5-18213, Invitrogen) was immobilized on tosyl-activated magnetic particles (MPs, Dynabeads M-450 Tosylactivated, Product no. 14203, Invitrogen). The tailored modification of the antibody on the MPs is described in detail in S2 (Supp. data). The anti-Legionella polyclonal antibody labelled with horseradish peroxidase (HRP) enzyme from rabbit was used as a secondary antibody (Catalogue no. PA1-73141, Invitrogen). The strains used were Legionella pneumophila (serogroup 1. Philadelphia 1, ATCC 33152), and for the specificity study, Pseudomonas aeruginosa (ATCC 15442), Klebsiella pneumoniae (Schroeter) Trevisan (ATCC BAA-1705), Mycobacterium fortuitum (ATCC 6841), Enterobacter spp. (used as control by Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica), Escherichia coli (ATCC 10536), and Salmonella choleraesuis (ATCC 13311).
The anti-L. pneumophila monoclonal antibody (G90A) from mouse (Catalogue no. MA5-18213, Invitrogen) was immobilized on tosyl-activated magnetic particles (MPs, Dynabeads M-450 Tosylactivated, Product no. 14203, Invitrogen). The tailored modification of the antibody on the MPs is described in detail in S
5
Enzyme Stabilization in Lyophilized Powder
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Lyophilized powder of peroxidase
from HRP, (type I, 89.63 U/mg solid, CAS 9003-99-0) was purchased
from Sigma-Aldrich (St. Louis, Missouri, USA) and used without further
purification. D-(+)-xylose (≥99%, CAS 58-86-6), glycerol (≥99.5%
CAS 56-81-5), D-sorbitol (≥98%, CAS 50-70-4), DL-proline (99%,
CAS 609-36-9), phenol-4-sulfonic acid sodium salt dihydrate (PSA,
98%, CAS 10580-19-5), 4-aminoantipyrine (4-AAP, ≥ 99%, CAS
83-07-8) and hydrogen peroxide 30% solution (CAS 7722-84-1) were purchased
from Sigma-Aldrich (St. Louis, Missouri, USA). Trehalose dihydrate
(CAS 6138-23-4) was kindly provided by Hayashibara Co., LDA (Okayama,
Japan). Betaine anhydrous (>97%, CAS 107-43-7) was obtained from
TCI
(Tokyo, Japan) and sucrose (CAS 57-50-1) was purchased from Cmd Chemicals
(Funchal, Portugal).
from HRP, (type I, 89.63 U/mg solid, CAS 9003-99-0) was purchased
from Sigma-Aldrich (St. Louis, Missouri, USA) and used without further
purification. D-(+)-xylose (≥99%, CAS 58-86-6), glycerol (≥99.5%
CAS 56-81-5), D-sorbitol (≥98%, CAS 50-70-4), DL-proline (99%,
CAS 609-36-9), phenol-4-sulfonic acid sodium salt dihydrate (PSA,
98%, CAS 10580-19-5), 4-aminoantipyrine (4-AAP, ≥ 99%, CAS
83-07-8) and hydrogen peroxide 30% solution (CAS 7722-84-1) were purchased
from Sigma-Aldrich (St. Louis, Missouri, USA). Trehalose dihydrate
(CAS 6138-23-4) was kindly provided by Hayashibara Co., LDA (Okayama,
Japan). Betaine anhydrous (>97%, CAS 107-43-7) was obtained from
TCI
(Tokyo, Japan) and sucrose (CAS 57-50-1) was purchased from Cmd Chemicals
(Funchal, Portugal).
6
Biopterin Synthesis and Characterization
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Tetrahydro-L-biopterin (BH4) hydrochloride and 7,8-dihydro-L-biopterin (BH2) hydrochloride were obtained from Cayman Chemical Company (Ann Arbor, MI, USA) (CAS: 69056-38-8). Hydrogen peroxide 30% solution (CAS: 7722-84-1), 1,4-dithioerythritol ≥ 99.0% (DTE) (CAS: 6892-68-8), diethylenetriaminepentaacetic acid 98% (DTPA) (CAS: 67-43-6), LPS from Escherichia coli (L-4005) and Salmonella typhosa (L6386), potassium dihydrogen phosphate (# P018.2) and phosphorus acid (# 9079.1) were obtained from Sigma, Merck KGaA, Darmstadt, Germany.
7
Colorimetric Detection of Cytochrome C Activity
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Tetraethylorthosilicate
(TEOS), CytC (from equine heart), 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), and Coomassie Brilliant Blue G-250 were
purchased from Sigma-Aldrich. Phosphoric acid and hydrogen peroxide
(30% solution) were purchased from EMD Millipore. HRP was purchased
from Gold Biotechnology. Guaiacol was purchased from Cayman Chemical
Company. Premium grade glass coverslips (25 mm × 25 mm ×
1 mm) were purchased from Fisher Scientific. All chemicals and materials
were used as received, with the exception of the glass coverslips,
which were cleaned prior to use. All UV–vis spectra were obtained
via a Shimadzu UV-2101PC UV–vis spectrometer.
(TEOS), CytC (from equine heart), 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), and Coomassie Brilliant Blue G-250 were
purchased from Sigma-Aldrich. Phosphoric acid and hydrogen peroxide
(30% solution) were purchased from EMD Millipore. HRP was purchased
from Gold Biotechnology. Guaiacol was purchased from Cayman Chemical
Company. Premium grade glass coverslips (25 mm × 25 mm ×
1 mm) were purchased from Fisher Scientific. All chemicals and materials
were used as received, with the exception of the glass coverslips,
which were cleaned prior to use. All UV–vis spectra were obtained
via a Shimadzu UV-2101PC UV–vis spectrometer.
8
Quantification of Methylglyoxal-Induced Modifications
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Methylglyoxal solution (40% w/v; M0252), 2-Deoxy-D -glucose (D8375), Aminoguanidine hydrochloride (396494), Hydrogen peroxide solution ≥ 30% (95302), Doxycycline hyclate (D9891) as well as Paraformaldehyde (16005) were purchased from Sigma-Aldrich. D(+)-Glucose was from Merck. Aprotinin was from Applichem, Pepstatin was from Pepta Nova GmbH, Leupetin was from Peptide Institute, Inc., HygromycinB was from InvivoGen, Nourseothricin (cloNAT) was from Werner BioAgents. Zymolyase 20T was purchased from Amsbio. DAPI solution was from Thermo Fisher scientific. The anti-MG-H1 antibody was generated as described previously [28] (link). The anti-GFP antibody was purchased from Roche (Cat. No. 11 814 460 001). The anti-actin antibody was from Millipore (clone C4, Cat. No. MAB1501). As secondary antibodies horseradish-linked goat anti-rat (Cell Signalling Technology, # 7077S) goat anti-mouse (Cell Signalling Technology, # 7076S) were used.
9
Quantitative Analysis of Colistin and Polymyxin B
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Colistin sulfate, polymixin B (PMB) sulfate, trichloroacetic acid (TCA), hydrogen peroxide solution 30% (w/w) in water, isopropyl alcohol and formic acid were all purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Acetonitrile (LC-MS grade) was purchased from Fisher Scientific International (Fairlawn, NJ, USA) and methanol (LC-MS grade) was purchased from VWR (Radnor, PA, USA). All chemicals and reagents were of high purity. Oasis HLB 1cc Vac cartridges, 30 mg sorbent per cartridge, 30 µm particle size (Oasis® HLB 1 cc, 30 mg), (Waters Assoc., Milford, MA, U.S.A.) were used in solid phase extraction procedures.
10
Enzymatic Oxidation with Laccase
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Hydrogen peroxide solution 30% (ACS reagent) and Laccase from Trametes versicolor (0.94 U/mg) were obtained from Sigma-Aldrich (Saint-Louis, MO, USA).
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